PARP1 inhibition prevents oxidative stress in age-related hearing loss via PAR-Ca2+-AIF axis in cochlear strial marginal cells.

Studies have highlighted oxidative damage in the inner ear as a critical pathological basis for sensorineural hearing loss, especially the presbycusis. Poly(ADP-ribose) polymerase-1 (PARP1) activation responds to oxidative stress-induced DNA damage with pro-repair and pro-death effects resembling two sides of the same coin. PARP1-related cell death, known as parthanatos, whose underlying mechanisms are attractive research hotspots but remain to be clarified. In this study, we observed that aged rats showed stria vascularis degeneration and oxidative damage, and PARP1-dependent cell death was prominent in age-related cochlear disorganization and dysfunction. Based on oxidative stress model of primary cultured stria marginal cells (MCs), we revealed that upregulated PARP1 and PAR (Poly(ADP-ribose)) polymers are responsible for MCs oxidative death with high mitochondrial permeability transition pore (mPTP) opening and mitochondrial membrane potential (MMP) collapse, while inhibition of PARP1 ameliorated the adverse outcomes. Importantly, the PARylation of apoptosis-inducing factor (AIF) is essential for its conformational change and translocation, which subsequently causes DNA break and cell death. Concretely, the interaction of PAR and truncated AIF (tAIF) is the mainstream in the parthanatos pathway. We also found that the effects of AIF cleavage and release were achieved through calpain activity and mPTP opening, both of which could be regulated by PARP1 via mediation of mitochondria Ca2+ concentration. In conclusion, the PAR-Ca2+-tAIF signaling pathway in parthanatos contributes to the oxidative stress damage observed in MCs. Targeting PAR-Ca2+-tAIF might be a potential therapeutic strategy for the early intervention of presbycusis and other oxidative stress-associated sensorineural deafness.

Synaptic vesicle protein 2A mitigates parthanatos via apoptosis-inducing factor in a rat model of pharmacoresistant epilepsy.

AIMS: Synaptic vesicle protein 2A (SV2A) is a unique therapeutic target for pharmacoresistant epilepsy (PRE). As seizure-induced neuronal programmed death, parthanatos was rarely reported in PRE. Apoptosis-inducing factor (AIF), which has been implicated in parthanatos, shares a common cytoprotective function with SV2A. We aimed to investigate whether parthanatos participates in PRE and is mitigated by SV2A via AIF. METHODS: An intraperitoneal injection of lithium chloride-pilocarpine was used to establish an epileptic rat model, and phenytoin and phenobarbital sodium were utilized to select PRE and pharmacosensitive rats. The expression of SV2A was manipulated via lentivirus delivery into the hippocampus. Video surveillance was used to assess epileptic ethology. Biochemical tests were employed to test hippocampal tissues following a successful SV2A infection. Molecular dynamic calculations were used to simulate the interaction between SV2A and AIF. RESULTS: Parthanatos core index, PARP1, PAR, nuclear AIF and MIF, γ-H2AX, and TUNEL staining were all increased in PRE. SV2A is bound to AIF to form a stable complex, successfully inhibiting AIF and MIF nuclear translocation and parthanatos and consequently mitigating spontaneous recurrent seizures in PRE. Moreover, parthanatos deteriorated after the SV2A reduction. SIGNIFICANCE: SV2A protected hippocampal neurons and mitigated epileptic seizures by inhibiting parthanatos via binding to AIF in PRE.

Experimental evidence for Parthanatos-like mode of cell death of heat-damaged human skin fibroblasts in a cell culture-based in vitro burn model.

The cellular mechanisms of burn conversion of heat damaged tissue are center of many studies. Even if the molecular mechanisms of heat-induced cell death are controversially discussed in the current literature, it is widely accepted that caspase-mediated apoptosis plays a central role. In the current study we wanted to develop further information on the nature of the mechanism of heat-induced cell death of fibroblasts in vitro. We found that heating of human fibroblast cultures (a 10 s rise from 37 °C to 67 °C followed by a 13 s cool down to 37 °C) resulted in the death of about 50% of the cells. However, the increase in cell death started with a delay, about one hour after exposure to heat, and reached the maximum after about five hours. The lack of clear evidence for an active involvement of effector caspase in the observed cell death mechanism and the lack of observation of the occurrence of hypodiploid nuclei contradict heat-induced cell death by caspase-mediated apoptosis. Moreover, a dominant heat-induced increase in PARP1 protein expression, which correlated with a time-delayed ATP synthesis inhibition, appearance of double-strand breaks and secondary necrosis, indicate a different type of cell death than apoptosis. Indeed, increased translocation of Apoptosis Inducing Factor (AIF) and Macrophage Migration Inhibitory Factor (MIF) into cell nuclei, which correlates with the mentioned enhanced PARP1 protein expression, indicate PARP1-induced, AIF-mediated and MIF-activated cell death. With regard to the molecular actors involved, the cellular processes and temporal sequences, the mode of cell death observed in our model is very similar to the cell death mechanism via Parthanatos described in the literature.

CHCHD4 binding affects the active site of apoptosis inducing factor (AIF): Structural determinants for allosteric regulation.

Apoptosis-inducing factor (AIF), which is confined to mitochondria of normal healthy cells, is the first identified caspase-independent cell death effector. Moreover, AIF is required for the optimal functioning of the respiratory chain machinery. Recent findings have revealed that AIF fulfills its pro-survival function by interacting with CHCHD4, a soluble mitochondrial protein which promotes the entrance and the oxidative folding of different proteins in the inner membrane space. Here, we report the crystal structure of the ternary complex involving the N-terminal 27-mer peptide of CHCHD4, NAD+, and AIF harboring its FAD (flavin adenine dinucleotide) prosthetic group in oxidized form. Combining this information with biophysical and biochemical data on the CHCHD4/AIF complex, we provide a detailed structural description of the interaction between the two proteins, validated by both chemical cross-linking mass spectrometry analysis and site-directed mutagenesis.

AIF translocation into nucleus caused by Aifm1 R450Q mutation: generation and characterization of a mouse model for AUNX1.

Mutations in AIFM1, encoding for apoptosis-inducing factor (AIF), cause AUNX1, an X-linked neurologic disorder with late-onset auditory neuropathy (AN) and peripheral neuropathy. Despite significant research on AIF, there are limited animal models with the disrupted AIFM1 representing the corresponding phenotype of human AUNX1, characterized by late-onset hearing loss and impaired auditory pathways. Here, we generated an Aifm1 p.R450Q knock-in mouse model (KI) based on the human AIFM1 p.R451Q mutation. Hemizygote KI male mice exhibited progressive hearing loss from P30 onward, with greater severity at P60 and stabilization until P210. Additionally, muscle atrophy was observed at P210. These phenotypic changes were accompanied by a gradual reduction in the number of spiral ganglion neuron cells (SGNs) at P30 and ribbons at P60, which coincided with the translocation of AIF into the nucleus starting from P21 and P30, respectively. The SGNs of KI mice at P210 displayed loss of cytomembrane integrity, abnormal nuclear morphology, and dendritic and axonal demyelination. Furthermore, the inner hair cells and myelin sheath displayed abnormal mitochondrial morphology, while fibroblasts from KI mice showed impaired mitochondrial function. In conclusion, we successfully generated a mouse model recapitulating AUNX1. Our findings indicate that disruption of Aifm1 induced the nuclear translocation of AIF, resulting in the impairment in the auditory pathway.

Cdc73 is a major regulator of apoptosis-inducing factor 1 expression in Saccharomyces cerevisiae via H3K36 methylation.

Apoptosis-inducing factor 1 (AIF1) overexpression is intimately linked to the sensitivity of yeast cells towards hydrogen peroxide or acetic acid. Therefore, studying the mechanism of AIF1 regulation in the cell would provide a significant understanding of the factors guiding yeast apoptosis. In this report, we show the time-dependent induction of AIF1 under hydrogen peroxide stress. Additionally, we find that AIF1 expression in response to hydrogen peroxide is mediated by two transcription factors, Yap5 (DNA binding) and Cdc73 (non-DNA binding). Furthermore, substituting the H3K36 residue with another amino acid significantly abrogates AIF1 expression. However, substituting the lysine (K) in H3K4 or H3K79 with alanine (A) does not affect AIF1 expression level under hydrogen peroxide stress. Altogether, reduced AIF1 expression in cdc73Δ is plausibly due to reduced H3K36me3 levels in the cells.

Autophagy inhibitors 3-MA and BAF may attenuate hippocampal neuronal necroptosis after global cerebral ischemia-reperfusion injury in male rats by inhibiting the interaction of the RIP3/AIF/CypA complex.

Our previous study found that receptor interacting protein 3 (RIP3) and apoptosis-inducing factor (AIF) were involved in neuronal programmed necrosis during global cerebral ischemia-reperfusion (I/R) injury. Here, we further studied its downstream mechanisms and the role of the autophagy inhibitors 3-methyladenine (3-MA) and bafilomycin A1 (BAF). A 20-min global cerebral I/R injury model was constructed using the 4-vessel occlusion (4-VO) method in male rats. 3-MA and BAF were injected into the lateral ventricle 1 h before ischemia. Spatial and activation changes of proteins were detected by immunofluorescence (IF), and protein interaction was determined by immunoprecipitation (IP). The phosphorylation of H2AX (γ-H2AX) and activation of mixed lineage kinase domain-like protein (p-MLKL) occurred as early as 6 h after reperfusion. RIP3, AIF, and cyclophilin A (CypA) in the neurons after I/R injury were spatially overlapped around and within the nucleus and combined with each other after reperfusion. The survival rate of CA1 neurons in the 3-MA and BAF groups was significantly higher than that in the I/R group. Autophagy was activated significantly after I/R injury, which was partially inhibited by 3-MA and BAF. Pretreatment with both 3-MA and BAF almost completely inhibited nuclear translocation, spatial overlap, and combination of RIP3, AIF, and CypA proteins. These findings suggest that after global cerebral I/R injury, RIP3, AIF, and CypA translocated into the nuclei and formed the DNA degradation complex RIP3/AIF/CypA in hippocampal CA1 neurons. Pretreatment with autophagy inhibitors could reduce neuronal necroptosis by preventing the formation of the RIP3/AIF/CypA complex and its nuclear translocation.

NADH improves AIF dimerization and inhibits apoptosis in iPSCs-derived neurons from patients with auditory neuropathy spectrum disorder.

Auditory neuropathy spectrum disorder (ANSD) is a hearing impairment involving disruptions to inner hair cells (IHCs), ribbon synapses, spiral ganglion neurons (SGNs), and/or the auditory nerve itself. The outcomes of cochlear implants (CI) for ANSD are variable and dependent on the location of lesion sites. Discovering a potential therapeutic agent for ANSD remains an urgent requirement. Here, 293T stable transfection cell lines and patient induced pluripotent stem cells (iPSCs)-derived auditory neurons carrying the apoptosis inducing factor (AIF) p.R422Q variant were used to pursue a therapeutic regent for ANSD. Nicotinamide adenine dinucleotide (NADH) is a main electron donor in the electron transport chain (ETC). In 293T stable transfection cells with the p.R422Q variant, NADH treatment improved AIF dimerization, rescued mitochondrial dysfunctions, and decreased cell apoptosis. The effects of NADH were further confirmed in patient iPSCs-derived neurons. The relative level of AIF dimers was increased to 150.7 % (P = 0.026) from 59.2 % in patient-neurons upon NADH treatment. Such increased AIF dimerization promoted the mitochondrial import of coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4), which further restored mitochondrial functions. Similarly, the content of mitochondrial calcium (mCa2+) was downregulated from 136.7 % to 102.3 % (P = 0.0024) in patient-neurons upon NADH treatment. Such decreased mCa2+ levels inhibited calpain activity, ultimately reducing the percentage of apoptotic cells from 30.5 % to 21.1 % (P = 0.021). We also compared the therapeutic effects of gene correction and NADH treatment on hereditary ANSD. NADH treatment had comparable restorative effects on functions of ANSD patient-specific cells to that of gene correction. Our findings offer evidence of the molecular mechanisms of ANSD and introduce NADH as a potential therapeutic agent for ANSD therapy.

Role of autophagy in stress and drug-responsive cell death in Entamoeba histolytica and its cross-talk with apoptosis-inducing factor.

Cell death in unicellular protozoan parasite Entamoeba histolytica is not yet reported though it displays several features of autophagic cell death. Autophagic cell death was reported to take place in ancient protozoans under several stresses. Here we report the occurrence of autophagic cell death in the Entamoeba histolytica trophozoites under oxidative stress as well as by the treatment with metronidazole, the most-widely-used drug for amoebiasis treatment and was shown to generate oxidative stress in the trophozoites. The autophagic flux increases during nutrient deprivation and metronidazole treatment and decreases upon oxidative stress. During oxidative stress the autophagy leads to nucleophagy that is ultimately destined to be digested within the lysosomal chamber. The formation of nucleophagosome depends on the apoptosis-inducing factor (AIF) that translocates to the nucleus from cytoplasm upon oxidative stress. It was experimentally proved that ATG8 (Autophagy-related protein 8) binds with the AIF in the nucleus of the trophozoites and helps in ATG8 recruitment and autophagy initiation overall suggesting that oxidative stress-driven AIF translocation to nucleus results in binding with ATG8 and initiates nucleophagy leading to cell death.

A Missense Variant in AIFM1 Caused Mitochondrial Dysfunction and Intolerance to Riboflavin Deficiency.

AIFM1 is a mitochondrial flavoprotein involved in caspase-independent cell death and regulation of respiratory chain complex biogenesis. Mutations in the AIFM1 gene have been associated with multiple clinical phenotypes, but the effectiveness of riboflavin treatment remains controversial. Furthermore, few studies explored the reasons underlying this controversy. We reported a 7-year-old boy with ataxia, sensorimotor neuropathy and muscle weakness. Genetic and histopathological analyses were conducted, along with assessments of mitochondrial function and apoptosis level induced by staurosporine. Riboflavin deficiency and supplementation experiments were performed using fibroblasts. A missense c.1019T > C (p. Met340Thr) variant of AIFM1 was detected in the proband, which caused reduced expression of AIFM1 protein and mitochondrial dysfunction as evidenced by downregulation of mitochondrial complex subunits, respiratory deficiency and collapse of ΔΨm. The proportion of apoptotic cells in mutant fibroblasts was lower than controls after induction of apoptosis. Riboflavin deficiency resulted in decreased AIFM1 protein levels, while supplementation with high concentrations of riboflavin partially increased AIFM1 protein levels in variant fibroblasts. In addition, mitochondrial respiratory function of mutant fibroblasts was partly improved after riboflavin supplementation. Our study elucidated the pathogenicity of the AIFM1 c.1019T > C variant and revealed mutant fibroblasts was intolerant to riboflavin deficiency. Riboflavin supplementation is helpful in maintaining the level of AIFM1 protein and mitochondrial respiratory function. Early riboflavin treatment may serve as a valuable attempt for patients with AIFM1 variant.

Impaired AIF-CHCHD4 interaction and mitochondrial calcium overload contribute to auditory neuropathy spectrum disorder in patient-iPSC-derived neurons with AIFM1 variant.

Auditory neuropathy spectrum disorder (ANSD) is a hearing impairment caused by dysfunction of inner hair cells, ribbon synapses, spiral ganglion neurons and/or the auditory nerve itself. Approximately 1/7000 newborns have abnormal auditory nerve function, accounting for 10%-14% of cases of permanent hearing loss in children. Although we previously identified the AIFM1 c.1265 G > A variant to be associated with ANSD, the mechanism by which ANSD is associated with AIFM1 is poorly understood. We generated induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells (PBMCs) via nucleofection with episomal plasmids. The patient-specific iPSCs were edited via CRISPR/Cas9 technology to generate gene-corrected isogenic iPSCs. These iPSCs were further differentiated into neurons via neural stem cells (NSCs). The pathogenic mechanism was explored in these neurons. In patient cells (PBMCs, iPSCs, and neurons), the AIFM1 c.1265 G > A variant caused a novel splicing variant (c.1267-1305del), resulting in AIF p.R422Q and p.423-435del proteins, which impaired AIF dimerization. Such impaired AIF dimerization then weakened the interaction between AIF and coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4). On the one hand, the mitochondrial import of ETC complex subunits was inhibited, subsequently leading to an increased ADP/ATP ratio and elevated ROS levels. On the other hand, MICU1-MICU2 heterodimerization was impaired, leading to mCa2+ overload. Calpain was activated by mCa2+ and subsequently cleaved AIF for its translocation into the nucleus, ultimately resulting in caspase-independent apoptosis. Interestingly, correction of the AIFM1 variant significantly restored the structure and function of AIF, further improving the physiological state of patient-specific iPSC-derived neurons. This study demonstrates that the AIFM1 variant is one of the molecular bases of ANSD. Mitochondrial dysfunction, especially mCa2+ overload, plays a prominent role in ANSD associated with AIFM1. Our findings help elucidate the mechanism of ANSD and may lead to the provision of novel therapies.

Apoptosis-inducing factor-like protein-mediated stress and metronidazole-responsive programmed cell death pathway in Entamoeba histolytica.

Apoptosis-inducing factor (AIF) is the major component of the caspase-independent cell death pathway that is considered to be evolutionarily ancient. Apoptosis is generally evolved with multicellularity as a prerequisite for the elimination of aged, stressed, or infected cells promoting the survival of the organism. Our study reports the presence of a putative AIF-like protein in Entamoeba histolytica, a caspase-deficient primitive protozoan, strengthening the concept of occurrence of apoptosis in unicellular organisms as well. The putative cytoplasmic EhAIF migrates to the nucleus on receiving stresses that precede its binding with DNA, following chromatin degradation and chromatin condensation as evident from both in vitro and in vivo experiments. Down-regulating the EhAIF expression attenuates the apoptotic features of insulted cells and increases the survival potency in terms of cell viability and vitality of the trophozoites, whereas over-expression of the EhAIF effectively enhances the phenomena. Interestingly, metronidazole, the most widely used drug for amoebiasis treatment, is also potent to elicit similar AIF-mediated cell death responses like other stresses indicating the AIF-mediated cell death could be the probable mechanism of trophozoite-death by metronidazole treatment. The occurrence of apoptosis in a unicellular organism is an interesting phenomenon that might signify the altruistic death that overall improves the population health.

Deep Brain Stimulation for the Management of AIFM1-Related Disabling Tremor: A Case Series.

The AIFM1 gene encodes a mitochondrial protein that acts as a flavin adenine dinucleotide-dependent nicotinamide adenine dinucleotide oxidase and apoptosis regulator. Monoallelic pathogenic AIFM1 variants result in a spectrum of X-linked neurological disorders, including Cowchock syndrome. Common features in Cowchock syndrome include a slowly progressive movement disorder, cerebellar ataxia, progressive sensorineural hearing loss, and sensory neuropathy. We identified a novel maternally inherited hemizygous missense AIFM1 variant, c.1369C>T p.(His457Tyr), in two brothers with clinical features consistent with Cowchock syndrome using next-generation sequencing. Both individuals had a progressive complex movement disorder phenotype, including disabling tremor poorly responsive to medications. Deep brain stimulation (DBS) of the ventral intermediate thalamic nucleus ameliorated contralateral tremor and improved their quality of life; this suggests the beneficial role for DBS in treatment-resistant tremor within AIFM1-related disorders.

p20BAP31 induces cell apoptosis via both AIF caspase-independent and the ROS/JNK mitochondrial pathway in colorectal cancer.

BACKGROUND: During cell apoptosis, the C-terminus of BAP31 is cleaved by caspase-8 and generates p20BAP31, which has been shown to induce an apoptotic pathway between the endoplasmic reticulum (ER) and mitochondria. However, the underlying mechanisms of p20BAP31 in cell apoptosis remains unclear. METHODS: We compared the effects of p20BAP31 on cell apoptosis in six cell lines and selected the most sensitive cells. Functional experiments were conducted, including Cell Counting Kit 8 (CCK-8), reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) assay. Then, cell cycle and apoptosis were investigated by flow cytometry and verified by immunoblotting. Next, NOX inhibitors (ML171 and apocynin), ROS scavenger (NAC), JNK inhibitor (SP600125), and caspase inhibitor (Z-VAD-FMK) were used to further investigate the underlying mechanisms of p20BAP31 on cell apoptosis. Finally, apoptosis-inducing factor (AIF) translocation from the mitochondria to the nuclei was verified by immunoblotting and immunofluorescence assay. RESULTS: We found that overexpression of p20BAP31 indeed induced apoptosis and had a much greater sensitivity in HCT116 cells. Furthermore, the overexpression of p20BAP31 inhibited cell proliferation by causing S phase arrest. Further study revealed that p20BAP31 reduced MMP, with a significant increase in ROS levels, accompanied by the activation of the MAPK signaling pathway. Importantly, the mechanistic investigation indicated that p20BAP31 induces mitochondrial-dependent apoptosis by activating the ROS/JNK signaling pathway and induces caspase-independent apoptosis by promoting the nuclear translocation of AIF. CONCLUSIONS: p20BAP31 induced cell apoptosis via both the ROS/JNK mitochondrial pathway and AIF caspase-independent pathway. Compared with antitumor drugs that are susceptible to drug resistance, p20BAP31 has unique advantages for tumor therapy.

Arsenite-induced cytotoxicity is regulated by poly-ADP ribose polymerase 1 activation and parthanatos in p53-deficient H1299 cells: The roles of autophagy and p53.

Arsenic is a double-edged sword metalloid since it is both an environmental carcinogen and a chemopreventive agent. Arsenic cytotoxicity can be dependent or independent of the tumor suppressor p53. However, the effects and the underlying molecular mechanisms of arsenic cytotoxicity in p53-deficient cells are still unclear. Here, we report a distinctive cell death mode via PARP-1 activation by arsenic in p53-deficient H1299 cells. H1299 (p53-/-) cells showed higher sensitivity to sodium arsenite (NaAR) than H460 (p53+/+) cells. H460 cells induced canonical apoptosis through caspase-dependent poly-ADP ribose polymerase 1 (PARP-1) cleavage and induced the expression of phospho-p53 and p21. However, H1299 cells induced poly-ADP-ribose (PAR) polymer accumulation and caspase-independent parthanatos, which was inhibited by 3-aminobenzamide (AB) and nicotinamide (NAM). Fractionation studies revealed the mitochondrial translocation of PAR polymers and nuclear translocation of the apoptosis-inducing factor (AIF). Although the exposure of NaAR to p53-overexpressing H1299 cells increased the PAR polymer levels, it inhibited parthanatos by inducing p21 and phospho-p53 expression. LC3-II and p62 accumulated in a NaAR dose- and exposure time-dependent manner, and this accumulation was further enhanced by autophagy inhibition, indicating that arsenic inhibits autophagic flux. p53 overexpression led to a decrease in the p62 levels, an increase in the LC3-II levels, and reduced parthanatos, indicating that arsenic induces p53-dependent functional autophagy. These results show that the NaAR-induced cytotoxicity in p53-deficient H1299 cells is regulated by PARP-1 activation-mediated parthanatos, which is promoted by autophagy inhibition. This suggests that PARP-1 activation could be used as an effective therapeutic approach for arsenic toxicity in p53-deficient cells.

AIFM1 variants associated with auditory neuropathy spectrum disorder cause apoptosis due to impaired apoptosis-inducing factor dimerization. / å¬ç¥žç»ç— ç›¸å ³AIFM1åŸºå› çªå˜å¼•èµ·å‡‹äº¡è¯±å¯¼å› å­äºŒèšä½“å½¢æˆéšœç¢å¹¶å¯¼è‡´ç»†èƒžå‡‹äº¡å¢žåŠ .

Auditory neuropathy spectrum disorder (ANSD) represents a variety of sensorineural deafness conditions characterized by abnormal inner hair cells and/or auditory nerve function, but with the preservation of outer hair cell function. ANSD represents up to 15% of individuals with hearing impairments. Through mutation screening, bioinformatic analysis and expression studies, we have previously identified several apoptosis-inducing factor (AIF) mitochondria-associated 1 (AIFM1) variants in ANSD families and in some other sporadic cases. Here, to elucidate the pathogenic mechanisms underlying each AIFM1 variant, we generated AIF-null cells using the clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system and constructed AIF-wild type (WT) and AIF-mutant (mut) (p.|T260A, p.|R422W, and p.|R451Q) stable transfection cell lines. We then analyzed AIF structure, coenzyme-binding affinity, apoptosis, and other aspects. Results revealed that these variants resulted in impaired dimerization, compromising AIF function. The reduction reaction of AIF variants had proceeded slower than that of AIF-WT. The average levels of AIF dimerization in AIF variant cells were only 34.5%|‒|49.7% of that of AIF-WT cells, resulting in caspase-independent apoptosis. The average percentage of apoptotic cells in the variants was 12.3%|‒|17.9%, which was significantly higher than that (6.9%|‒|7.4%) in controls. However, nicotinamide adenine dinucleotide (NADH) treatment promoted the reduction of apoptosis by rescuing AIF dimerization in AIF variant cells. Our findings show that the impairment of AIF dimerization by AIFM1 variants causes apoptosis contributing to ANSD, and introduce NADH as a potential drug for ANSD treatment. Our results help elucidate the mechanisms of ANSD and may lead to the provision of novel therapies.

SIRT5-related desuccinylation modification of AIFM1 protects against compression-induced intervertebral disc degeneration by regulating mitochondrial homeostasis.

Mitochondrial dysfunction plays a major role in the development of intervertebral disc degeneration (IDD). Sirtuin 5 (SIRT5) participates in the maintenance of mitochondrial homeostasis through its desuccinylase activity. However, it is still unclear whether succinylation or SIRT5 is involved in the impairment of mitochondria and development of IDD induced by excessive mechanical stress. Our 4D label-free quantitative proteomic results showed decreased expression of the desuccinylase SIRT5 in rat nucleus pulposus (NP) tissues under mechanical loading. Overexpression of Sirt5 effectively alleviated, whereas knockdown of Sirt5 aggravated, the apoptosis and dysfunction of NP cells under mechanical stress, consistent with the more severe IDD phenotype of Sirt5 KO mice than wild-type mice that underwent lumbar spine instability (LSI) surgery. Moreover, immunoprecipitation-coupled mass spectrometry (IP-MS) results suggested that AIFM1 was a downstream target of SIRT5, which was verified by a Co-IP assay. We further demonstrated that reduced SIRT5 expression resulted in the increased succinylation of AIFM1, which in turn abolished the interaction between AIFM1 and CHCHD4 and thus led to the reduced electron transfer chain (ETC) complex subunits in NP cells. Reduced ETC complex subunits resulted in mitochondrial dysfunction and the subsequent occurrence of IDD under mechanical stress. Finally, we validated the efficacy of treatments targeting disrupted mitochondrial protein importation by upregulating SIRT5 expression or methylene blue (MB) administration in the compression-induced rat IDD model. In conclusion, our study provides new insights into the occurrence and development of IDD and offers promising therapeutic approaches for IDD.

The natural product dehydrocurvularin induces apoptosis of gastric cancer cells by activating PARP-1 and caspase-3.

The natural product dehydrocurvularin (DSE2) is a fungal-derived macrolide with potent anticancer activity, but the mechanism is still unclear. We found that DSE2 effectively inhibited the growth of gastric cancer cells and induced the apoptosis by activating Poly(ADP-ribose) polymerase 1 (PARP-1) and caspase-3. Pharmacological inhibition and genetic knockdown with PARP-1 or caspase-3 suppressed DSE2-induced apoptosis. PARP-1 was previously reported to be cleaved into fragments during apoptosis. However, PARP-1 was barely cleaved in DSE2-induced apoptosis. DSE2 induced PARP-1 activation as indicated by rapid depletion of NAD+ and the concomitant formation of poly(ADP-ribosylated) proteins (PARs). Interestingly, the PARP-1 inhibitor (Olaparib) attenuated the cytotoxicity of DSE2. Moreover, the combination of Olaparib and Z-DEVD-FMK (caspase-3 inhibitor) further reduced the cytotoxicity. It has been shown that PARP-1 activation triggers cytoplasm-nucleus translocation of apoptosis-inducing factor (AIF). Caspase-3 inhibitors inhibited PARP-1 activation and suppressed PARP-1-induced AIF nuclear translocation. These results indicated that DSE2-induced caspase-3 activation may occur before PARP-1 activation. The ROS inhibitor, N-acetyl-cysteine, significantly inhibited the activation of caspase-3 and PARP-1, indicating that ROS overproduction contributed to DSE2-induced apoptosis. Using an in vivo approach, we further found that DSE2 significantly inhibited gastric tumor growth and promoted translocation of AIF to the nucleus. In conclusion, DSE2 induces gastric cell apoptosis by activating caspase-3 and PARP-1, and shows potent antitumor activity against human gastric carcinoma in vitro and in vivo.

Programmed mechanisms of status epilepticus-induced neuronal necrosis.

Excitotoxicity is the underlying mechanism for all acute neuronal injury, from cerebral ischemia, status epilepticus, traumatic CNS injury, and hypoglycemia. It causes morphological neuronal necrosis, and it triggers a programmed cell death program. Excessive calcium entry through the NMDA-receptor-operated cation channel activates two key enzymes-calpain I and neuronal nitric oxide synthase (nNOS). Calpain I, a cytosolic enzyme, translocates to mitochondrial and lysosomal membranes, causing release of cytochrome c, endonuclease G, and apoptosis-inducing factor (AIF) from mitochondria and DNase II and cathepsins B and D from lysosomes. These all translocate to neuronal nuclei, creating DNA damage, which activates poly(ADP) ribose polymerase-1 (PARP-1) to form excessive amounts of poly(ADP) ribose (PAR) polymers, which translocate to mitochondrial membranes, causing release of truncated AIF (tAIF). The free radicals that are released from mitochondria and peroxynitrite, formed from nitric oxide (NO) from nNOS catalysis of L-arginine to L-citrulline, damage mitochondrial and lysosomal membranes and DNA. The end result is the necrotic death of neurons. Another programmed necrotic pathway, necroptosis, occurs through a parallel pathway. As investigators of necroptosis do not recognize the excitotoxic pathway, it is unclear to what extent each contributes to programmed neuronal necrosis. We are studying the extent to which each contributes to acute neuronal necrosis and the extent of cross-talk between these pathways.